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The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature, Infectious Virions.

Gershburg S, Geltz J, Peterson KE, Halford WP, Gershburg E - PLoS ONE (2015)

Bottom Line: Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread.Loss of UL13 function alone also had no appreciable effects on viral DNA levels.These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL 62794-9626, United States of America.

ABSTRACT
Herpes simplex virus type 1 (HSV-1) encodes two bona fide serine/threonine protein kinases, the US3 and UL13 gene products. HSV-1 ΔUS3 mutants replicate with wild-type efficiency in cultured cells, and HSV-1 ΔUL13 mutants exhibit <10-fold reduction in infectious viral titers. Given these modest phenotypes, it remains unclear how the US3 and UL13 protein kinases contribute to HSV-1 replication. In the current study, we designed a panel of HSV-1 mutants, in which portions of UL13 and US3 genes were replaced by expression cassettes encoding mCherry protein or green fluorescent protein (GFP), respectively, and analyzed DNA replication, protein expression, and spread of these mutants in several cell types. Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread. Loss of UL13 function alone also had no appreciable effects on viral DNA levels. However, loss of UL13 function did result in a measurable decrease in the steady-state levels of two viral glycoproteins (gC and gD), release of total and infectious virions, and viral spread. Disruption of both genes did not affect the accumulation of viral DNA, but resulted in further reduction in gC and gD steady-state levels, and attenuation of viral spread and infectious virion release. These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release. Moreover, the data suggest that the combined activities of the US3 and UL13 protein kinases are critical to the efficient assembly and release of infectious virions from HSV-1-infected cells.

No MeSH data available.


Related in: MedlinePlus

HSV-1 kinase mutants are attenuated in different cell types.Monolayers of (A) Vero, (B) SK-N-SH, and (C) HFF-1 cells were inoculated with wild type HSV-1 KOS or HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants at an MOI of 0.1 pfu per cell and incubated for 24 hours. Virus yields were determined by plaque assay on Vero cells. Results are presented as the mean virus yield ± sem (n = 6; ***—p<0.001).
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pone.0131420.g008: HSV-1 kinase mutants are attenuated in different cell types.Monolayers of (A) Vero, (B) SK-N-SH, and (C) HFF-1 cells were inoculated with wild type HSV-1 KOS or HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants at an MOI of 0.1 pfu per cell and incubated for 24 hours. Virus yields were determined by plaque assay on Vero cells. Results are presented as the mean virus yield ± sem (n = 6; ***—p<0.001).

Mentions: To determine whether the effects of the loss of UL13 and US3 kinases on HSV-1 replication is cell-type-dependent, infectious virus production was verified in two additional cell types; namely, the human neuroblastoma SK-N-SH cell line (Fig 8B) and HFF-1 human foreskin fibroblasts (Fig 8C), while Vero cells served as a control (Fig 8A). Monolayers of cells were inoculated with 0.1 pfu per cell of HSV-1 KOS, or HSV-1 ΔUL13, ΔUS3, and ΔUL13/ΔUS3 mutants and virus-infected cells were freeze-thawed at 24 hours post-inoculation to quantify viral titers in cell lysates. In Vero cells, wild-type HSV-1 KOS produced titers of 5.8 ± 0.1 log (pfu/ml) and the HSV-1 ΔUS3 mutant produced equivalent titers of 5.9 ± 0.3 log (pfu/ml) (Fig 8A). The HSV-1 ΔUL13 mutant produced titers of 5.2 ± 0.3 log (pfu/ml), which were slightly lower than wild-type HSV-1 KOS (Fig 8A). Consistent with its micro-plaque phenotype, the HSV-1 ΔUL13/ΔUS3 mutant produced the lowest titers of infectious virus on Vero cells, 4.8 ± 0.1 log (pfu/ml), which were ~12-fold lower than those produced by HSV-1 KOS-infected cells (Fig 8A). In SK-N-SH cells, the HSV-1 ΔUS3 or ΔUL13 mutant viruses produced 5- and 4-fold lower titers of infectious virus relative to wild-type HSV-1, respectively, whereas the HSV-1 ΔUL13/ΔUS3 mutant virus produced on average 30-fold lower titer of infectious virus (Fig 8B). In the HFF-1 cells, loss of function of either US3 or UL13 kinase significantly impaired the production of infectious virus. Specifically, in HFF-1 cells, the HSV-1 ΔUS3 mutant and ΔUL13 mutant produced 13- and ~370-fold lower titers of infectious virus relative to wild-type HSV-1 (Fig 8C). Loss of US3 and UL13 kinase function had a combined effect resulting in ~2,750-fold reduction in titers of infectious virus relative to wild-type HSV-1 (Fig 8C).


The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature, Infectious Virions.

Gershburg S, Geltz J, Peterson KE, Halford WP, Gershburg E - PLoS ONE (2015)

HSV-1 kinase mutants are attenuated in different cell types.Monolayers of (A) Vero, (B) SK-N-SH, and (C) HFF-1 cells were inoculated with wild type HSV-1 KOS or HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants at an MOI of 0.1 pfu per cell and incubated for 24 hours. Virus yields were determined by plaque assay on Vero cells. Results are presented as the mean virus yield ± sem (n = 6; ***—p<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482649&req=5

pone.0131420.g008: HSV-1 kinase mutants are attenuated in different cell types.Monolayers of (A) Vero, (B) SK-N-SH, and (C) HFF-1 cells were inoculated with wild type HSV-1 KOS or HSV-1 ΔUS3, ΔUL13, and ΔUL13/ΔUS3 mutants at an MOI of 0.1 pfu per cell and incubated for 24 hours. Virus yields were determined by plaque assay on Vero cells. Results are presented as the mean virus yield ± sem (n = 6; ***—p<0.001).
Mentions: To determine whether the effects of the loss of UL13 and US3 kinases on HSV-1 replication is cell-type-dependent, infectious virus production was verified in two additional cell types; namely, the human neuroblastoma SK-N-SH cell line (Fig 8B) and HFF-1 human foreskin fibroblasts (Fig 8C), while Vero cells served as a control (Fig 8A). Monolayers of cells were inoculated with 0.1 pfu per cell of HSV-1 KOS, or HSV-1 ΔUL13, ΔUS3, and ΔUL13/ΔUS3 mutants and virus-infected cells were freeze-thawed at 24 hours post-inoculation to quantify viral titers in cell lysates. In Vero cells, wild-type HSV-1 KOS produced titers of 5.8 ± 0.1 log (pfu/ml) and the HSV-1 ΔUS3 mutant produced equivalent titers of 5.9 ± 0.3 log (pfu/ml) (Fig 8A). The HSV-1 ΔUL13 mutant produced titers of 5.2 ± 0.3 log (pfu/ml), which were slightly lower than wild-type HSV-1 KOS (Fig 8A). Consistent with its micro-plaque phenotype, the HSV-1 ΔUL13/ΔUS3 mutant produced the lowest titers of infectious virus on Vero cells, 4.8 ± 0.1 log (pfu/ml), which were ~12-fold lower than those produced by HSV-1 KOS-infected cells (Fig 8A). In SK-N-SH cells, the HSV-1 ΔUS3 or ΔUL13 mutant viruses produced 5- and 4-fold lower titers of infectious virus relative to wild-type HSV-1, respectively, whereas the HSV-1 ΔUL13/ΔUS3 mutant virus produced on average 30-fold lower titer of infectious virus (Fig 8B). In the HFF-1 cells, loss of function of either US3 or UL13 kinase significantly impaired the production of infectious virus. Specifically, in HFF-1 cells, the HSV-1 ΔUS3 mutant and ΔUL13 mutant produced 13- and ~370-fold lower titers of infectious virus relative to wild-type HSV-1 (Fig 8C). Loss of US3 and UL13 kinase function had a combined effect resulting in ~2,750-fold reduction in titers of infectious virus relative to wild-type HSV-1 (Fig 8C).

Bottom Line: Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread.Loss of UL13 function alone also had no appreciable effects on viral DNA levels.These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL 62794-9626, United States of America.

ABSTRACT
Herpes simplex virus type 1 (HSV-1) encodes two bona fide serine/threonine protein kinases, the US3 and UL13 gene products. HSV-1 ΔUS3 mutants replicate with wild-type efficiency in cultured cells, and HSV-1 ΔUL13 mutants exhibit <10-fold reduction in infectious viral titers. Given these modest phenotypes, it remains unclear how the US3 and UL13 protein kinases contribute to HSV-1 replication. In the current study, we designed a panel of HSV-1 mutants, in which portions of UL13 and US3 genes were replaced by expression cassettes encoding mCherry protein or green fluorescent protein (GFP), respectively, and analyzed DNA replication, protein expression, and spread of these mutants in several cell types. Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread. Loss of UL13 function alone also had no appreciable effects on viral DNA levels. However, loss of UL13 function did result in a measurable decrease in the steady-state levels of two viral glycoproteins (gC and gD), release of total and infectious virions, and viral spread. Disruption of both genes did not affect the accumulation of viral DNA, but resulted in further reduction in gC and gD steady-state levels, and attenuation of viral spread and infectious virion release. These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release. Moreover, the data suggest that the combined activities of the US3 and UL13 protein kinases are critical to the efficient assembly and release of infectious virions from HSV-1-infected cells.

No MeSH data available.


Related in: MedlinePlus